Regulation of the Melanocyte Lineage by the AP2 Transcription Factor Family

干细胞维持的缺陷会导致组织再生能力的丧失，或者相反， 在生长失调和癌症中的作用干细胞如何抵抗失去其再生品质，同时保留 对扩散控制的了解仍然很少。黑素细胞干细胞（MSC）是研究黑素细胞分化的理想模型。 成体干细胞的研究。这种细胞类型的凋亡或分化可以导致毛发 变灰和MITF突变，MITF编码黑素细胞发育的主要调节因子， 导致老鼠毛发变白。TFAP 2A（转录因子激活增强子结合）突变 蛋白2 α）引起鳃-眼-面综合征（BOFS），其中包括过早的头发变白。 然而，TFAP 2蛋白对MSC中相关调控网络的作用机制 仍然不清楚。基于斑马鱼、小鼠和培养的人类黑素细胞的大量初步数据， 我们的总体假设是AP-2活性调节黑素细胞的多能性、生长和分化 和MSC，与MITF平行工作并由KCTD 15介导的类小泛素化调节。在目标1中， 测试由两个旁系同源物冗余提供的细胞自主TFAP 2活性， 这是维持MSC所必需的，并且BOFS相关的TFAP 2A变体抑制这两种旁系同源物， 导致MSC的异位分化和头发变灰。我们将在小鼠中测试这一假设， MSC或MSC龛的细胞中的相关Tfap 2旁系同源物，并检查毛发变灰和MSC状态。 在目的2中，我们将检验TFAP 2旁系同源物在基因座的子集处充当先锋因子以 MITF与之结合，促进MITF介导的促进生长和分化的基因活化。这 将需要从黑色素瘤细胞系中删除TFAP 2旁系同源物，然后使用ATAC-评估开放染色质 SEQ的MITF的结合，以及使用ChIP-SEQ.我们将在原代成黑素细胞中进行平行实验 分离自斑马鱼胚胎。在目标3中，我们将检验TFAP 2A中的负反馈回路 信号传导对于MSC的维持和募集的正确平衡是必需的。我们发现一个 TFAP 2-parabolic反式激活活性的有效抑制剂KCTD 15在黑素细胞中表达， 斑马鱼kctd 15 a/B突变体表现出多余的异位黑素细胞。我们将测试Kctd 15- 介导的类小泛素化阻断了Tfap 2a的反式激活活性，在体外测试类小泛素化。我们将测试 kctd 15 a通过消除其在黑素细胞中的表达在控制黑素细胞分化中的作用 脉我们还将测试TFAP 2抑制介导KCTD 15在细胞中的作用的预测。 黑素细胞谱系拟议研究的预期成果是对途径有更深入的了解 调节MSC生长和分化的平衡。对这些机制的理解 MITF和TFAP 2蛋白有助于MSC的维持，以及这些因子是如何调节的， 广泛的影响，因为它们参与衰老机制和黑色素瘤的发生和发展。

Defects in the maintenance of stem cells can result in loss of the ability of tissues to regenerate or, conversely, in dysregulated growth and cancer. How the stem cell resists losing its regenerative qualities while retaining control of proliferation remains poorly understood. The melanocyte stem cell (MSC) is an ideal model for the study of adult stem cells in general. Either the apoptosis or differentiation of this cell type can cause hair graying, and mutations in MITF, which encodes the master regulator of melanocyte development, have been shown to cause hair graying in mice. Mutations in TFAP2A (Transcription Factor Activating-enhancer binding Protein 2 alpha) cause branchio-oculo-facial syndrome (BOFS), which includes premature hair graying. However, the mechanisms whereby TFAP2 proteins contribute to the relevant regulatory networks in MSCs remain unclear. Based on extensive preliminary data from zebrafish, mice, and cultured human melanocytes, our overall hypothesis is that AP-2 activity regulates pluripotency, growth and differentiation in melanocytes and MSCs, working in parallel with MITF and regulated by KCTD15-mediated sumoylation. In Aim 1 we will test the hypothesis that that cell-autonomous TFAP2 activity, provided redundantly by two paralogs, is necessary for the maintenance of MSCs, and that BOFS-associated TFAP2A variants inhibit both paralogs, leading to ectopic differentiation of MSCs and to hair graying. We will test this hypothesis in mice by deleting the relevant Tfap2 paralogs in MSCs or in cells of the MSC niche, and examining hair graying and MSC status. In Aim 2 we will test the hypothesis that TFAP2 paralogs serve as pioneer factors at a subset of the loci to which MITF binds, facilitating MITF-mediated activation of genes that promote growth and differentiation. This will entail deleting TFAP2 paralogs from a melanoma cell line and then assessing open chromatin using ATAC- SEQ, and binding of MITF using ChIP-SEQ. We will carry out parallel experiments in primary melanoblasts isolated from zebrafish embryos. In Aim 3 we will test the hypothesis that a negative feedback loop in TFAP2A signaling is necessary for the correct balance of maintenance and recruitment of MSCs. We found that a potent inhibitor of TFAP2-paralog transactivation activity, KCTD15, is expressed in melanocytes, and that zebrafish kctd15a/b mutants exhibit supernumerary, ectopic melanocytes. We will test the model that Kctd15- mediated sumoylation of Tfap2a blocks its transactivation activity, testing sumoylation in vitro. We will test kctd15a for a role in controlling melanocyte differentiation by abrogating its expression in the melanocyte lineage. We will also test the prediction that inhibition of TFAP2 mediates the effects of KCTD15 in the melanocyte lineage. The expected outcome of the proposed research is a deeper understanding of pathways that regulate the balance of growth and differentiation of MSCs. An understanding of the mechanisms whereby MITF and TFAP2 proteins contribute to MSC maintenance, and of how these factors are regulated, will have a broad impact as they participate in mechanisms of aging and in the initiation and progression of melanoma.